8 Questions You May Have About Cryopreserving Bovine In Vivo–Derived Embryos

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Published on: December 27, 2017

John F. Hasler
Cell: 970-222-5302

Dr. Pat Comyn, the new chair of the AETA Education Committee, asked me to write a short piece clarifying some issues concerning the cryopreservation of bovine embryos for inclusion in the December issue of A Closer Look. The AETA has come a long way since our humble beginnings in 1983, and our 2017 membership now totals 556, including a large increase in the number of new members. The following facts and suggestions will be of most interest to our new and less experienced members. Not only are there many variables involved in successfully freezing and thawing bovine embryos, there also are many variations on most of the steps that do not notably detract from success rates. Having worked with many ET practitioners in 17 different states and a number of foreign countries, I have a pretty good idea of what works well and what does not. The following points are either based on published data that I deem to be replicable or based on my own experience and observations. Please feel free to contact me should you want advice or clarification.


  1. What embryos should be chosen for freezing? Based on a pregnancy data set from five large ET businesses, totaling almost 73,000 embryos frozen in EG (ethylene glycol) and then thawed and transferred, it was possible to very accurately determine the success rate of different embryo stages. In this data set, stages 4 and 5 were virtually identical with a 55% pregnancy rate, whereas stage 6 embryos were slightly, but highly significantly (P < 0.001), lower at 52% and stage 7 lower yet, at 44% (P < 0.001). The 3–percentage point decrease with stage 6 embryos is pretty trivial commercially, but the loss of 11 percentage points with stage 7 (expanded blastocysts) is quite large. However, I would always choose to freeze this stage if no recipients were available, but also notify the owner to expect a decreased pregnancy rate. These data should encourage ET practitioners to try avoiding day-8 flushes whenever possible, unless the embryos are to be transferred fresh.


  1. How long and under what conditions should embryos be held between the time of flushing and freezing? My own data indicate that there was no decrease in pregnancy rate when embryos were held for up to 180 minutes in a standard holding medium at room temperature. A fairly recent study in France indicated that there was no decrease in viability for embryos held up to five hours. Beyond that, I would anticipate some loss of viability.


  1. What cryopreservation medium should I use? A number of businesses manufacture and market media that are reliable. Obviously, the overwhelming majority of bovine embryos are now frozen in media containing EG as the cryoprotectant. Glycerol, however, is still used on a small scale, and although it involves more time and effort at the time of thawing, it is very reliable. I do not know of any data indicating that there is any difference in efficacy between EG with or without a small concentration of sucrose, although based on sales records from Vetoquinol Inc., EG + sucrose is the most popular. There is a formulation variation on EG freezing media termed SynGro Freeze, manufactured by Vetoquinol Inc.* Results with this medium are comparable to traditional media containing EG, the notable difference being that SynGro does not need refrigeration for shipment or storage.


  1. How should straws be labelled? Labelling should include all the information mandated by the IETS, including the practitioner’s freeze code identification number. Also, straw colors must correspond with the origin of the embryos (in vivo, in vitro, cloned, and so on). Labelling should not be applied directly to the straw containing the embryo, and it is highly recommended that a label printer be used to ensure that the information is legible.


  1. Guidelines for loading straws—The embryo should be isolated toward the middle of the straw within a column of freezing medium and with an air space separating it from additional fluid columns above and below. I prefer two short columns behind the embryo column and one column extending to the open end of the straw in front of embryo. When filling the straw, it is important to draw the medium up fairly rapidly, just before the straw is filled, in order for the first medium column to penetrate the inner wick column and fully saturate the polyvinyl chloride. This greatly decreases the chances of liquid nitrogen entering the straw on that end. There are many methods of sealing the open end of the straw. I prefer using the tapered one quarter/one half milliliter plugs, which allow a 0.5-mL straw to be used for the label, which can be easily identified and read without exposing the 0.25-mL straw containing the embryo to the air.


  1. Why must I seed the straws? The most important principle involved in successfully freezing embryos is to remove enough water and provide a cryoprotectant to prevent formation of ice crystals within the cells. The actual freezing temperature of the solutions commonly used to freeze bovine embryos (EG at 10% and glycerol at 1.4 M) is just slightly below −3°C. However, due to the phenomenon known as supercooling, these media can often be cooled to −12°C or so before solidifying. Seeding, or starting the formation of ice within the media surrounding the embryo, initially involves the freezing of only pure water, while pockets of liquid, with increasing solute osmolarity, continue to draw water out of the cells. If the embryo is not seeded, then this dehydration does not occur, and when the temperature reaches

−12°C or so, freezing of the embryo will suddenly occur without the initial dehydration. This will usually be fatal when the embryo is thawed. Programmable embryo freezers are usually held at −5 to −7°C while the straws are loaded and then placed in the freezer. After a minute or so at this temperature, the straws can be seeded by touching a column of medium either below or above (or both) the embryo column with a metal spatula, Q tip, and so on that has been submerged in liquid nitrogen. The column will freeze and the ice will move down the thin layer of media coating the wall of the straw around the air space and then initiate freezing of the embryo column. It is strongly suggested that all straws be checked a minute after seeding to ensure that the seeds have held. In addition, it is recommended that the seeding temperature be maintained for 10 minutes or so before the temperature of the freezer starts decreasing. Because the PVA surfactant in SynGro does not always reliably coat the straw wall around the air space, it is recommended that seeding be initiated at the lower end of air space just above the embryo column.


  1. At what rate of decline should embryos be frozen after seeding? For many years, most of the ET industry used a freezing rate of 0.3°C per minute. The practice today is faster, in the range of 0.5 or 0.6°C per minute. The results are excellent and the freezing is completed in a shorter period of time. It is recommended that embryos be cooled to about −32°C, with the freezer holding at that temperature. Embryos can be removed from the freezer when it reaches −32°C or held at that temperature for up to 30 minutes or so. Embryos must be removed from the freezer with minimum exposure time to air, immediately plunged into liquid nitrogen, and then loaded into storage goblets in a deep Dewar of nitrogen or a foam box. Embryos must be stored submerged in liquid nitrogen or, for a limited time, in a fully charged nitrogen “vapor” tank. Embryos should not be exposed to air when reading labels and moving them between tanks.


  1. How do I deal with programmable freezer problems? A detailed discussion of this subject is beyond the scope of this discussion. Over the years I have been called innumerable times regarding problems involving the mechanics and electronics of programmable freezers. Some of the problems were not really serious, but they ranged from minor to what was undoubtedly a fatal outcome, with many cases on a “maybe” or “I’m not sure” basis. I am happy to continue providing advice on freezer problems, so feel free to call me.


*Please note that although I currently am employed as a technical consultant by Vetoquinol Inc., I am not exclusively endorsing their media to the exclusion of products manufactured by other media companies.


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